The in vivo function of a protein is improved by binding it to a physiologically acceptable polymer molecule. In particular, binding a physiologically active protein to a physiologically acceptable polymer molecule has been found to substantially prolong its in vivo half-life. For example, U.S. Pat. No. 4,970,300 describes that the conjugation of a physiologically acceptable polymer molecule to factor VIII results in a factor VIII protein being activable by thrombin and having a substantially decreased antigenicity and immunoreactivity and a substantially increased in vivo disappearance time in the bloodstream of a mammal.
U.S. Pat. No. 4,970,300 describes that the conjugation of a polymer molecule (dextran) to Factor VIII (FVIII) results in a FVIII protein activatable by thrombin, and having a substantially decreased antigenicity and immunoreactivity and a substantially increased in vivo retention time in the bloodstream of a mammal. International patent application WO 94/15625 describes that conjugating factor VIII to a physiologically acceptable polymer molecule improves the in vivo function of factor VIII (i) by increasing its resistance to in vivo hydrolysis and thus prolonging its activity after administration, (ii) by significantly prolonging its circulating life in vivo over unmodified protein, and (iii) by increasing its absorption time into the blood stream. U.S. Pat. No. 6,037,452 describes FVIII and Factor IX (FIX) conjugates, where the protein is covalently bound to a poly(alkylene oxide) through carbonyl-groups in the protein. Further, improving the in vivo function of factor IX by binding it to physiologically acceptable polymer molecules, in particular poly(ethylene glycol) (“PEG”), has been described in international patent application WO 94/29370. A PEGylated FVIII that retains specific activity was disclosed in International Patent Publication WO/2007/126808. The conjugation of physiologically acceptable polymer to an active agent such as a protein is performed by preparing stable polymer-protein conjugates or polymer-protein conjugates in which the physiologically acceptable polymer is attached to the protein via releasable covalent bonds (pro-drug concept), i.e. a hydrolyzable or releaseable linker. For example, a releasable PEG moiety has been developed using a 9-flourenemethoxycarbonyl (FMOC) conjugation system containing two PEG chains (Nektar Inc., Huntsville Ala.). In addition an N-hydroxysuccinimide ester (NHS) group, which is useful for the chemical modification of lysine residues of the protein, may be linked to the fluorene ring system via the methoxycarbonyl group to generate the releasable PEG moiety. International Patent Publication WO 2008/082669 (incorporated herein by reference) describes a series of PEGylated recombinant FVIII variants based on the releasable PEG concept.
However, at present no reliable method for the quantitative determination of physiologically acceptable polymer molecules bound to proteins or nanoparticles is available apart from insensitive colorimetric methods (Nag et al. 1997, Anal Biochem 250:35-43), which allow only an estimation of the content of physiologically acceptable polymer molecules. Moreover, monoclonal antibodies for the determination of PEG concentrations have been disclosed (U.S. Pat. No. 6,617,118), but so far no system is available for the reliable determination of the amount of physiologically acceptable polymer molecule bound to a protein.
Therefore, a need exists for a new system to determine the amount of a physiologically acceptable polymer molecule, in particular PEG, bound to a protein, particularly a physiologically active protein.